B cell lineage acute lymphoblastic leukemia (ALL) represents by far the most frequent type of malignancy in children and teenagers. Despite significant advances in ALL treatment, many patients still die because of drug- resistance or leukemia relapse. Since recent work implicated leukemia stem cells in both drug-resistance and relapse of the disease, current therapy approaches focus on leukemia stem cell eradication. In contrast to B cell lineage ALL, leukemia stem cells in myeloid lineage leukemia have been extensively characterized. Acute and chronic myeloid leukemias develop hierarchically from a phenotypically distinct stem cell population. However, recent work suggests that no such hierarchy exists in B cell lineage ALL, which precludes an unequivocal phenotypic definition of leukemia stem cells in ALL. Therefore, we propose to functionally characterize leukemia stem cells in B cell lineage ALL based on an operational definition, i.e. the ability to maintain stem cell self-renewal and overcome oncogene-induced senescence. Specifically, we will test the hypothesis that the BCL6 transcriptional repressor represents one of the critical factors in B cell lineage ALL to maintain a pool of functional leukemia stem cells. The BCL6 proto-oncogene is frequently translocated in diffuse large B cell lymphoma (DLBCL) and maintains self-renewal capacity of DLBCL cells by transcriptional repression of p53 in the lymphoma cells. However, an oncogenic function of BCL6 has not been described in other cell types so far. In preliminary studies for this proposal, we have discovered aberrant expression of BCL6 as a central component of a fundamentally novel pathway of leukemia stem cell maintenance: BCL6 prevents leukemia stem cell depletion through negative regulation of the Arf/p53/p21 pathway in B cell lineage ALL. Compared to leukemias from BCL6-/- mice, BCL6 is required for the reactivation of an early embryonic gene expression program, development of drug-resistance, leukemia cell colony formation and leukemia-initiation in serially transplanted NOD/SCID mice in B cell lineage but not myeloid lineage leukemia. A novel retro-inverso BCL6 peptide inhibitor (RI-BPI) strongly synergized with tyrosine kinase inhibitors in the treatment of B cell lineage ALL cells in vitro and in vivo. This proposal will test the hypothesis that BCL6-dependent self-renewal represents a critical requirement for stem cell maintenance in B cell lineage ALL. Based on the discovery of BCL6 as a key component of a fundamentally novel pathway of stem cell self-renewal in various subtypes of ALL, we propose three Aims to develop these findings towards application in patient care: (1) To test the hypothesis that aberrant expression of BCL6 in leukemia cells prevents Arf/p53-mediated senescence and promotes stem cell quiescence, (2) To identify transcriptional targets of BCL6 in primary human leukemia cells and their contribution to self-renewal signaling and (3) To validate BCL6-BPI as a therapy adjuvant for targeted eradication of leukemia stem cells.